Project Summary/Abstract A fundamental question in neuroscience and human health is how do different brain states alter neuronal connections and how are these changes carried out at the cellular and molecular levels? We propose to address this question by examining neuronal connections through out the compact, completely described nervous system of the transparent nematode C. elegans as its whole brain activity cycles between wakefulness and sleep. Specifically, we propose to use our ability to modulate the C. elegans brain state to examine how the structure and function of excitatory as well as inhibitory synapses are changed as a function of recurrent neural activity. Further, we will identify the molecular mechanisms by which this is achieved. In Aim 1, We will ask how the brain states affect synaptic architecture across the animal's nervous system by testing different types of connections throughout the animals for their response to wakefulness and sleep. We will then ask whether the number or size of connections is affected. We will also identify the molecular regulators of the brain state-dependent synaptic changes, and visualize synaptic components in each brain state to determine how and when synapses are altered. These studies would provide the first evidence of broad sleep-dependent synaptic remodeling in C. elegans. In Aim 2, We will characterize the activity calcium transients (GCaMP6/7), cGMP fluxes (WincG) and neuropeptide-driven GPCR ligand binding (D-lite adapted reporters) of the entire brain of C. elegans as it sleeps and compare that to the wakeful animal. Using this information we will assess the pattern of these changes and relate them to the structure of synaptic components within the units that change most. We will attempt to understand if the synaptic changes are dispersed brain-wide and whether these connections are responsible for stabilizing sleep dependent changes in behavior. We will then alter the brain activity using optical manipulation of ChR, Arch, our cGMP-sponge WincD and cell and timing specific regulation of neuropeptide processing during sleep to test the requirement for patterned activity to direct changes in connections that we observe. In this way, we will begin to understand how brain state effects structural changes that affect behavior. These studies would provide the first brain wide understanding of the interplay between the activity and structure of connections in any animal. This understanding will provide insights into novel approaches to therapies aimed at mitigating activity-driven changes in human brain activity that promote maladaptive responses to activity such as addiction and post traumatic stress disorder.